US5703049A - High methionine derivatives of α-hordothionin for pathogen-control - Google Patents
High methionine derivatives of α-hordothionin for pathogen-control Download PDFInfo
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- US5703049A US5703049A US08/608,786 US60878696A US5703049A US 5703049 A US5703049 A US 5703049A US 60878696 A US60878696 A US 60878696A US 5703049 A US5703049 A US 5703049A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Definitions
- This invention relates to the improvement of feed formulations. Specifically, this invention relates to derivatives of ⁇ -hordothionin which provide higher percentages of the essential amino acid methionine in plants, while retaining the anti-pathogenic functionality of hordothionins.
- Feed formulations are required to provide animals essential nutrients critical to growth.
- crop plants are generally rendered food sources of poor nutritional quality because they contain low proportions of several amino acids which are essential for, but cannot be synthesized by, animals.
- One alternative method is to express a heterologous protein of favorable amino acid composition at levels sufficient to obviate food or feed supplementation.
- a number of seed proteins rich in sulfur amino acids have been identified.
- a key to good expression of such proteins involves efficient expression cassettes with seed specific promoters. Not only must the gene-controlling regions direct the synthesis of high levels of mRNA, the mRNA must be translated into stable protein.
- a second method of controlling pathogens has been the use of biological control organisms which are typically natural competitors or inhibitors of the pathogens.
- biological control organisms which are typically natural competitors or inhibitors of the pathogens.
- it is difficult to apply biological control organisms to large areas, and even more difficult to cause the living organisms to remain in the treated area for an extended period.
- a third method is the use of recombinant DNA techniques to insert cloned genes into plant cells wherein the genes express anti-microbial compounds.
- This technology has given rise to additional concerns about eventual microbial resistance to well-known, naturally occurring anti-microbials, particularly in the face of heavy selection pressure, which may occur in some areas.
- a continuing effort is under way to express naturally occurring anti-microbial compounds in plant cells directly by translation of a single structural gene.
- ⁇ -hordothionins can be modified to enhance their content of methionine while maintaining their anti-microbial activity.
- hordothionin derivatives can be expressed to simultaneously enhance both resistance to pathogenic diseases and methionine content of the plant.
- ⁇ -hordothionin is a 45-amino acid protein which has been well characterized. It can be isolated from seeds of barley (Hordeum vulgare). The molecule is stabilized by four disulfide bonds resulting from eight cysteine residues.
- the amino acid sequence is as provided in SEQUENCE I.D. No. 1. It has powerful anti-microbial properties. In its native form, the protein is especially rich in arginine and lysine residues, containing 5 residues (10%) of each. However, it is devoid of the essential amino acid methionine.
- the protein has been synthesized and the three-dimensional structure determined by computer modeling.
- the modeling of the protein predicts that the ten charged residues (arginine at positions 5,10,17,19 and 30, and lysine at positions 1,23,32,38 and 45) all occur on the surface of the molecule.
- the side chains of the polar amino acids (asparagine at position 11, glutamine at position 22 and threonine at position 41) also occur on the surface of the molecule.
- the hydrophobic amino acids (such as the side chains of leucine at positions 8,15,24 and 33 and valine at position 18) are also solvent-accessible.
- Three-dimensional modeling of the protein indicates that the arginine residue at position 10 is critical to retention of the appropriate 3-dimensional structure and possible folding through hydrogen bond interactions with the C-terminal residue of the protein.
- a methionine substitution at that point would disrupt the hydrogen bonding involving arginine at position 10, serine at position 2 and lysine at position 45, leading to a destabilization of the structure.
- the synthetic peptide having this substitution could not be made to fold correctly, which supported this analysis.
- Conservation of the arginine residue at position 10 provided a protein which folded correctly.
- methionine is a hydrophobic amino acid
- the resulting compound has the sequence indicated in SEQUENCE I.D. No. 2.
- the molecule is synthesized by solid phase peptide synthesis and folds into a stable structure. It has seven methoinine residues (15.5%) and, including the eight cysteines, the modified protein has a sulfur amino acid content of 33%.
- SEQUENCE I.D. No. 2 is illustrative of the present invention, it is not intended to be a limitation. Methionine substitutions can also be performed at positions containing charged amino acids. Only arginine at position 10 is critical for maintaining the structure of the protein through a hydrogen-bonding network with serine at position 2 and lysine at position 45. Thus, one can substitute methionine for lysine at positions 1,23,32, and/or 38, and for arginine at positions 5,17,19 and/or 30. The resulting compound has the sequence indicated in SEQUENCE I.D. No. 3.
- each peptide was confirmed by automated edman degradation on an applied biosystems 477a protein sequencer/120a pth analyzer. More preferably, however, the compounds of this invention are synthesized in vivo by bacterial or plant cells which have been transformed by insertion of an expression cassette containing a synthetic gene which when transcribed and translated yields the desired compound.
- an expression cassette containing a synthetic gene which when transcribed and translated yields the desired compound.
- Such empty expression cassettes providing appropriate regulatory sequences for plant or bacterial expression of the desired sequence, are also well-known, and the nucleotide sequence for the synthetic gene, either RNA or DNA, can readily be derived from the amino acid sequence for the protein using standard reference texts.
- such synthetic genes will employ plant-preferred codons to enhance expression of the desired protein.
- compositions of this invention and the methods of making and using them.
- other methods known by those of ordinary skill in the art to be equivalent, can also be employed.
- the polypeptides employed in this invention can be effectively applied to plants afflicted with susceptible microorganisms by any convenient means, including spray, creams, dust or other formulation common to the anti-microbial arts.
- the compound can also be incorporated systematically into the tissues of a treated plant so that in the course of infesting the plant the pathogens will be exposed to anti-microbial amounts of a compound of this invention.
- One method of doing this is to incorporate the compound in a non-phytotoxic vehicle which is adapted for systemic administration to the susceptible plants. This method is commonly employed with fungicidal materials such as captan and is well within the purview of one of ordinary skill in the art of plant fungicide formulation.
- an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA sequence coding for a compound of this invention in proper reading frame, together with transcription initiator and promoter sequences active in the plant. Transcription and translation of the DNA sequence under control of the regulatory sequences causes expression of the protein sequence at levels which provide an anti-microbial amount of the protein in the tissues of the plant which are normally infected by the pathogens.
- the plant is preferably a plant susceptible to infection and damage by one or more of Fusarium graminearum, Fusarium moniliforme, Aspergillus flavus, Alternaria longipes, Sclerotinia sclerotiorum, and Sclerotina trifoliorum.
- Fusarium graminearum Fusarium moniliforme
- Aspergillus flavus Alternaria longipes
- Sclerotinia sclerotiorum Sclerotina trifoliorum
- These include corn (Zea mays) and sorghum (Sorghum bicolor).
- this is not to be construed as limiting, inasmuch as these two species are among the most difficult commercial crops to reliably transform and regenerate, and these pathogens also infect certain other crops.
- the methods of this invention are readily applicable via conventional techniques to numerous plant species, if they are found to be susceptible to the plant pathogens listed hereinabove, including, without limitation, species from the genera Allium, Antirrhinum, Arabidopsis, Arachis, Asparagus, Atropa, Arena, Beta, Brassica, Browallia, Capsicum, Cicer, Cicla, Citrullus, Citrus, Cucumis, Cucurbita, Datura daucus, Digitalis, Fagopyrum, Fragaria, Geranium, Glycine, Gossypium, Helianthus, Hordeum, Hemerocallis, Lactuca, Lens, Lolium, Lotus, Lycopersicon, Majorana, Manihot, Medicago, Nasturtium, Nicotiana, Oryza, Pelargonium, Persea, Petunia, Phaseolus, Pisum, Ranunculus, Raphanus, Ricinus, Saccharum, Secale, Senecio, Set
- genes which code for the present compounds can be inserted into an appropriate expression cassette and introduced into cells of a plant species.
- an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA sequence coding for a compound of this invention in proper reading frame, together with transcription initiator and promoter sequences active in the plant. Transcription and translation of the DNA sequence under control of the regulatory sequences causes expression of the protein sequence at levels which provide an elevated amount of the protein in the tissues of the plant.
- Preferred plants that are to be transformed according to the methods of this invention are cereal crops, including maize, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean.
- Synthetic DNA sequences can then be prepared which code for the appropriate sequence of amino acids, and this synthetic DNA sequence can be inserted into an appropriate plant expression cassette.
- expression cassette is meant a complete set of control sequences including initiation, promoter and termination sequences which function in a plant cell when they flank a structural gene in the proper reading frame.
- Expression cassettes frequently and preferably contain an assortment of restriction sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence.
- the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA.
- a preferred (empty) expression cassette into which the cDNA of the present invention can be inserted is the pPHI414 plasmid developed by Beach, et al., of Pioneer Hi-Bred International, Inc., Johnston, IA, as disclosed in U.S. patent application No. 07/785,648; (1991); incorporated herein in its entirety by reference.
- Highly preferred plant expression cassettes will be designed to include one or more selectable marker genes, such as kanamycin resistance or herbicide tolerance genes.
- vector herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell.
- the vector has one or more endonuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme.
- Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as markers to identify and separate transformed cells.
- Preferred markers/selection agents include kanamycin, chlorosulfuron, phosphonothricin, hygromycin and methotrexate.
- a cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant.”
- a particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule which is not a part of the chromosomes of the cell.
- genomic and cDNA encoding the gene of interest may be used in this invention.
- the vector of interest may also be constructed partially from a cDNA clone and partially from a genomic clone.
- genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene in the host cell.
- the genetic construct will contain (a) a first genetic sequence coding for the protein or trait of interest and (b) one or more regulatory sequences operably linked on either side of the structural gene of interest.
- the regulatory sequences will be selected from the group comprising of promoters and terminators.
- the regulatory sequences may be from autologous or heterologous sources.
- Promoters that may be used in the genetic sequence include NOS, OCS and CaMV promoters.
- An efficient plant promoter that may be used is an overproducing plant promoter.
- Overproducing plant promoters that may be used in this invention include the promoter of the cholorophyll ⁇ - ⁇ binding protein, and the promoter of the small sub-unit (ss) of the ribulose-1,5-biphosphate carboxylase from soybean.
- ss small sub-unit
- These two promoters are known to be light-induced, in eukaryotic plant cells. See e.g., An Agricultural Perspective, A. Cashmore, Pelham, New York, 1983, pp. 29-38, G.
- the expression cassette comprising the structural gene for the protein of this invention operably linked to the desired control sequences can be ligated into a suitable cloning vector.
- plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used.
- the cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells. Typically, genes conferring resistance to antibiotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers.
- an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector.
- the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells.
- Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S. typhimurium, and Serratia marcescens.
- Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention. Since these hosts are also microorganisms, it will be essential to ensure that plant promoters which do not cause expression of the protein in bacteria are used in the vector.
- the isolated cloning vector will then be introduced into the plant cell using any convenient technique, including electroporation (in protoplasts), retroviruses, bombardment, and microinjection into cells from monocotyledonous or dicotyledonous plants in cell or tissue culture to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette.
- the monocotyledonous species will be selected from maize, sorghum, wheat or rice, and the dicotyledonous species will be selected from soybean, alfalfa, rapeseed, sunflower or tomato.
- protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the gene for a protein according to this invention.
- a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette of this invention.
- this invention provides methods of imparting resistance to diseases caused by microorganisms selected from Fusarium graminearum, Fusarium moniliforme, Diplodia maydis, Collectototrichum graminicola, Verticillium alboatrum, Phytophthora megaspermae f.sp. glycinea, Macrophomina phaseolina, Diaporthe phaseolorum caulivora, Sclerotinia sclerotiorum, Sclerotinia trifoliorum, Aspergillus falvus to plants of a susceptible taxon, comprising the steps of:
- taxon herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varieties, variants, and other minor taxonomic groups which lack a consistent nomenclature.
- pathogen means any organism, bacterial or fungal, capable of causing disease in plants.
- anti-pathogenic or anti-microbial activity means activity to prevent and/or combat and/or alleviate infection by a pathogen.
- anti-microbial amount herein is meant an amount of either polypeptide or combination thereof sufficient to provide anti-microbial activity so as to alleviate or prevent infection by susceptible organisms in the plant at a reasonable benefit/risk ratio.
- the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous species.
- this invention provides a method for increasing methionine levels in Agrobacterium tumefaciens-susceptible dicotyledonous plants in which the expression cassette is introduced into the cells by infecting the cells with agrobacterium tumefaciens, a plasmid of which has been modified to include a plant expression cassette of this invention.
Abstract
Description
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 3 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: LysSerCysCysArgSerThrLeuGlyArgAsnCysTyrAsnLeuCys 151015 ArgValArgGlyAlaGlnLysLeuCysAlaGlyValCysArgCysLys 202530 LeuThrSerSerGlyLysCysProThrGlyPheProLys 354045 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: LysSerCysCysArgSerThrMetGlyArgMetCysTyrAsnMetCys 151015 ArgMetArgGlyAlaMetLysLeuCysAlaGlyValCysArgCysLys 202530 MetThrSerSerGlyLysCysProMetGlyPheProLys 354045 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: MetSerCysCysMetSerThrMetGlyArgMetCysTyrAsnMetCys 151015 MetMetMetGlyAlaMetMetMetCysAlaGlyValCysMetCysMet 202530 MetThrSerSerGlyMetCysProMetGlyPheProLys 354045 __________________________________________________________________________
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